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Biochemistry and Cell Biology =... Jun 2024Sperm nuclear basic proteins (SNBPs) were isolated from extracted antheridia-rich male gametophytes raised from spores of the swordfern, . Electrophoretic (acetic...
Sperm nuclear basic proteins (SNBPs) were isolated from extracted antheridia-rich male gametophytes raised from spores of the swordfern, . Electrophoretic (acetic acid-urea PAGE and SDS-PAGE) and chromatographic (rp-HPLC) characterization of the nuclear proteins exhibited the characteristics of the histone (H-type). In both types of gel electrophoresis, histones H1, H2A, and H2B showed an altered electrophoretic mobility corresponding to that which is routinely observed for the histones in other plants. Histones present during spermatogenesis of the fern were compared with the few current SNBPs known to be present in higher and lower evolutionary plant clades. A transition from an early protamine (P-type) SNBPs in charophytes and bryophytes to the (H-type) SNBP observed here is reminiscent of similar reversions observed in the animal kingdom.
Topics: Ferns; Plant Proteins; Nuclear Proteins; Histones; Amino Acid Sequence; Electrophoresis, Polyacrylamide Gel; Molecular Sequence Data
PubMed: 38346284
DOI: 10.1139/bcb-2023-0343 -
Scientific Reports Feb 2024Spermatogenesis is one of the most dramatic changes in cell differentiation. Remarkable chromatin condensation of the nucleus is observed in animal, plant, and algal...
Spermatogenesis is one of the most dramatic changes in cell differentiation. Remarkable chromatin condensation of the nucleus is observed in animal, plant, and algal sperm. Sperm nuclear basic proteins (SNBPs), such as protamine and sperm-specific histone, are involved in chromatin condensation of the sperm nucleus. Among brown algae, sperm of the oogamous Fucales algae have a condensed nucleus. However, the existence of sperm-specific SNBPs in Fucales algae was unclear. Here, we identified linker histone (histone H1) proteins in the sperm and analyzed changes in their gene expression pattern during spermatogenesis in Sargassum horneri. A search of transcriptomic data for histone H1 genes in showed six histone H1 genes, which we named ShH1.1a, ShH1b, ShH1.2, ShH1.3, ShH1.4, and ShH1.5. Analysis of SNBPs using SDS-PAGE and LC-MS/MS showed that sperm nuclei contain histone ShH1.2, ShH1.3, and ShH1.4 in addition to core histones. Both ShH1.2 and ShH1.3 genes were expressed in the vegetative thallus and the male and female receptacles (the organs producing antheridium or oogonium). Meanwhile, the ShH1.4 gene was expressed in the male receptacle but not in the vegetative thallus and female receptacles. From these results, ShH1.4 may be a sperm-specific histone H1 of S. horneri.
Topics: Animals; Male; Histones; Sargassum; Chromatography, Liquid; Semen; Tandem Mass Spectrometry; Cell Nucleus; Chromatin; Spermatozoa
PubMed: 38336896
DOI: 10.1038/s41598-024-53729-2 -
ACS Omega Jan 2024Insulin NPH is an intermediate-acting insulin. Its protracted action profile is due to the formation of microcrystalline suspensions when insulin is complexed with a...
Insulin NPH is an intermediate-acting insulin. Its protracted action profile is due to the formation of microcrystalline suspensions when insulin is complexed with a basic peptide protamine, zinc ion, and phenolic ligands. Despite advancements in analytical techniques, the binding epitope and binding mode of the protamine in the insulin-protamine complex are still unknown. In this study, we used bioinformatics tools such as molecular docking and molecular dynamics (MD) simulations to compute the binding sites and energetics of the insulin-protamine complex. We have taken four naturally occurring protamine peptides that are independently docked with the insulin R6 hexamer and subjected them to 200 ns MD simulations to observe the dynamics of the complexes and estimate the binding energies. The arginine-rich protamine peptides were found to bind on the surface of the insulin hexamer through hydrogen bonding, hydrophobic, and electrostatic interactions well supported by the calculated negative binding energies. The overall structure of the insulin hexamer was retained upon binding, highlighting its dynamic stability in the complex. Furthermore, the residues at the termini of the protamine peptides in the complex were seen to be highly dynamic, which stabilize toward the end of the simulation.
PubMed: 38313521
DOI: 10.1021/acsomega.3c08445 -
JBRA Assisted Reproduction Jun 2024Male infertility is a great matter of concern as out of 15% of infertile couples in the reproductive age, about 40% are contributed by male factors alone. For DNA... (Review)
Review
Male infertility is a great matter of concern as out of 15% of infertile couples in the reproductive age, about 40% are contributed by male factors alone. For DNA condensation during spermatogenesis, constrained DNA nicking is required, which if increased beyond certain level results in infertility in men. High sperm DNA Fragmentation (SDF) majorly contributes to male infertility and its association with regards to poor natural conception and assisted reproductive technology (ART) outcomes is equivocal. Apoptosis, protamination failure and the excess of reactive oxygen species (ROS) are considered to be the main causes of SDF. It's testing came into existence because of the limitations of the conventional methods in explaining infertility in normozoospermic infertile individuals. Over the past 25 years, SDF's several testing strategies have been proposed to diagnose the aetiology of infertility. Various treatments combined with sperm selection techniques are being used alone or in combination to reduce DNA fragmentation index (DFI) and obtain spermatozoa with high quality chromatin for assisted reproduction. This review summarises SDF's main causes, its impact on fertility and clinical outcomes in assisted reproduction, the need to perform test, testing procedures, and the treatment strategies.
Topics: Humans; Male; DNA Fragmentation; Infertility, Male; Spermatozoa; Reproductive Techniques, Assisted
PubMed: 38289201
DOI: 10.5935/1518-0557.20230076 -
JA Clinical Reports Jan 2024This study investigated whether intraoperative blood salvage was associated with coagulation disorder diagnosed by conventional coagulation tests and thromboelastography...
BACKGROUND
This study investigated whether intraoperative blood salvage was associated with coagulation disorder diagnosed by conventional coagulation tests and thromboelastography (TEG) after cardiopulmonary bypass (CPB).
STUDY DESIGN AND METHODS
This was a prospective, observational study. Ninety-two patients who underwent cardiovascular surgery with CPB were enrolled. We evaluated coagulation function in patients with or without cell salvage blood transfusion at the following time points: before CPB, just after protamine administration, and 1 h after protamine administration. We evaluated platelet count, fibrinogen concentration, and TEG parameters. Patients were considered to have coagulation disorder if one or more of the following criteria were present: (1) residual heparin, (2) low platelet count, (3) low fibrinogen level, (4) low clotting factor level, and (5) hyperfibrinolysis.
RESULTS
Fifty-three of 92 patients (57.6%) received intraoperative cell salvage. Coagulation disorder was observed in 56 of 92 patients (60.9%) after CPB. There was no significant difference between patients with or without intraoperative blood salvage in terms of the incidence of coagulation disorder (p = 0.542) or the total volume of blood from the drain after CPB (p = 0.437). Intraoperative blood salvage was not associated with coagulation disorder diagnosed by either TEG or conventional coagulation tests (odds ratio 1.329, 95% confidence interval: 0.549-3.213, p = 0.547). There were no significant interactions between patients with or without intraoperative blood salvage regarding coagulation parameters derived from TEG.
CONCLUSIONS
The incidence of coagulation disorder and the total blood volume from the drain after CPB did not differ significantly between patients with or without intraoperative blood salvage.
PubMed: 38270666
DOI: 10.1186/s40981-024-00689-1 -
Frontiers in Immunology 2023In the tumor microenvironment (TME), tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer....
BACKGROUND
In the tumor microenvironment (TME), tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer. Toll-like receptors (TLRs) ligands, such as poly(I:C) or resiquimod (R848) are able to reprogram TAMs towards M1-like antitumor effector cells. The objective of our work has been to develop and evaluate polymeric nanocapsules (NCs) loaded with poly(I:C)+R848, to improve drug stability and systemic toxicity, and evaluate their targeting and therapeutic activity towards TAMs in the TME of solid tumors.
METHODS
NCs were developed by the solvent displacement and layer-by-layer methodologies and characterized by dynamic light scattering and nanoparticle tracking analysis. Hyaluronic acid (HA) was chemically functionalized with mannose for the coating of the NCs to target TAMs. NCs loaded with TLR ligands were evaluated for toxicity and immunostimulatory activity by Alamar Blue, ELISA and flow cytometry, using primary human monocyte-derived macrophages. For experiments, the CMT167 lung cancer model and the MN/MCA1 fibrosarcoma model metastasizing to lungs were used; tumor-infiltrating leukocytes were evaluated by flow cytometry and multispectral immunophenotyping.
RESULTS
We have developed polymeric NCs loaded with poly(I:C)+R848. Among a series of 5 lead prototypes, protamine-NCs were selected based on their physicochemical properties (size, charge, stability) and characterization, showing good biocompatibility on primary macrophages and ability to stimulate their production of T-cell attracting chemokines (CXCL10, CCL5) and to induce M1-like macrophages cytotoxicity towards tumor cells. In mouse tumor models, the intratumoral injection of poly(I:C)+R848-protamine-NCs significantly prevented tumor growth and lung metastasis. In an orthotopic murine lung cancer model, the intravenous administration of poly(I:C)+R848-prot-NCs, coated with an additional layer of HA-mannose to improve TAM-targeting, resulted in good antitumoral efficacy with no apparent systemic toxicity. While no significant alterations were observed in T cell numbers (CD8, CD4 or Treg), TAM-reprogramming in treated mice was confirmed by the relative decrease of interstitial alveolar macrophages, having higher CD86 expression but lower CD206 and Arg1 expression in the same cells, in treated mice.
CONCLUSION
Mannose-HA-protamine-NCs loaded with poly(I:C)+R848 successfully reprogram TAMs , and reduce tumor progression and metastasis spread in mouse tumors.
Topics: Humans; Animals; Mice; Tumor-Associated Macrophages; Nanocapsules; Mannose; Lung Neoplasms; Disease Models, Animal; Protamines; Tumor Microenvironment; Imidazoles
PubMed: 38259462
DOI: 10.3389/fimmu.2023.1334800 -
International Journal of Molecular... Jan 2024Infertility affects millions of couples worldwide and has a profound impact not only on their families, but also on communities. Telomere attrition has been associated...
Infertility affects millions of couples worldwide and has a profound impact not only on their families, but also on communities. Telomere attrition has been associated with infertility, DNA damage and fragmentation. Oxidative stress has been shown to affect sperm DNA integrity and telomere length. Sirtuins such as SIRT1 and SIRT3 are involved in aging and oxidative stress response. The aim of the present study is to determine the role of SIRT1 and SIRT3 in regulating oxidative stress, telomere shortening, and their association with oligospermia. Therefore, we assessed the protein levels of SIRT1 and SIRT3, total antioxidant capacity (TAC), superoxide dismutase (SOD), malondialdehyde (MDA) and catalase activity (CAT) in the seminal plasma of 272 patients with oligospermia and 251 fertile men. We also measured sperm telomere length (STL) and leukocyte telomere length (LTL) using a standard real-time quantitative PCR assay. Sperm chromatin and protamine deficiency were also measured as per standard methods. Our results for oligospermic patients demonstrate significant reductions in semen parameters, shorter STL and LTL, lower levels of SOD, TAC, CAT, SIRT1 and SIRT3 levels, and also significant protamine deficiency and higher levels of MDA and DNA fragmentation. We conclude that a shorter TL in sperms and leukocytes is associated with increased oxidative stress that also accounts for high levels of DNA fragmentation in sperms. Our results support the hypothesis that various sperm parameters in the state of oligospermia are associated with or caused by reduced levels of SIRT1 and SIRT3 proteins.
Topics: Humans; Male; Semen; Oligospermia; Antioxidants; Sirtuin 3; Sirtuin 1; Spermatozoa; Protamines; Superoxide Dismutase
PubMed: 38255792
DOI: 10.3390/ijms25020718 -
Molecular Medicine (Cambridge, Mass.) Jan 2024Molecular techniques can complement conventional spermiogram analyses to provide new information on the fertilizing potential of spermatozoa and to identify early...
BACKGROUND
Molecular techniques can complement conventional spermiogram analyses to provide new information on the fertilizing potential of spermatozoa and to identify early alterations due to environmental pollution.
METHODS
Here, we present a multilevel molecular profiling by small RNA sequencing and sperm nuclear basic protein analysis of male germ cells from 33 healthy young subjects residing in low and high-polluted areas.
RESULTS
Although sperm motility and sperm concentration were comparable between samples from the two sites, those from the high-pollution area had a higher concentration of immature/immune cells, a lower protamine/histone ratio, a reduced ability of sperm nuclear basic proteins to protect DNA from oxidative damage, and an altered copper/zinc ratio in sperm. Sperm levels of 32 microRNAs involved in intraflagellar transport, oxidative stress response, and spermatogenesis were different between the two areas. In parallel, a decrease of Piwi-interacting RNA levels was observed in samples from the high-polluted area.
CONCLUSIONS
This comprehensive analysis provides new insights into pollution-driven epigenetic alterations in sperm not detectable by spermiogram.
Topics: Male; Humans; RNA, Small Untranslated; Semen; Sperm Motility; Spermatozoa; Environment; Nuclear Proteins
PubMed: 38243211
DOI: 10.1186/s10020-023-00776-6 -
Molecules (Basel, Switzerland) Jan 2024Protamine is a cationic peptide derived from fish sperm and has several important functional properties: antibacterial properties, acting as a carrier for injectable...
Protamine is a cationic peptide derived from fish sperm and has several important functional properties: antibacterial properties, acting as a carrier for injectable insulin and as a heparin antagonist, combatting fatigue, etc. Thus, it has been widely used in medicinal applications and food products. Cultured is a type of pufferfish with a delicious taste that is popular in China, and its production is increasing significantly. Therefore, protamine was extracted via acid extraction from the sperm of and further isolated and purified via sephadex gel chromatography, ion exchange chromatography, and desalination chromatography. Furthermore, the physicochemical properties of protamine were investigated. The results showed that the sperm of the cultured were non-toxic, and the extracted and purified protamine had high contents of arginine (36.90%) and lysine (27.02%), respectively. The secondary structure of protamine was mainly β-folded and irregularly curled. Additionally, protamine exhibited high thermal stability with a denaturation temperature of 176 °C. This study would provide a theoretical basis for the structural analysis, bioactivity, and resource development of pufferfish protamine and help to promote the development of the pufferfish industry.
Topics: Male; Animals; Takifugu; Protamines; Semen; Heparin Antagonists; Anti-Bacterial Agents
PubMed: 38202846
DOI: 10.3390/molecules29010263 -
Research and Practice in Thrombosis and... Nov 2023Anti-Xa assays are used for unfractionated heparin (UFH) monitoring. Dextran sulfate (DS) is used in some assays to overcome the artifactual preanalytical release of...
BACKGROUND
Anti-Xa assays are used for unfractionated heparin (UFH) monitoring. Dextran sulfate (DS) is used in some assays to overcome the artifactual preanalytical release of platelet factor 4. However, the practical implications of this test modification have not been studied extensively.
OBJECTIVES
To investigate the impact of the presence of DS in the anti-Xa assay for UFH laboratory monitoring.
METHODS
We studied factor Xa inhibition, using an assay without DS (Stago Liquid Anti-Xa), in normal pool plasma spiked with various concentrations of UFH (up to 1 IU/mL) in the presence of increasing concentrations of DS (up to 2560 μg/mL). We also investigated the effect of DS on FXa inhibition measured after the addition of UFH and heparin antagonists (protamine and Polybrene; Sigma Aldrich). Eventually, we compared the anti-Xa levels measured using the assay without DS to those measured with an assay containing DS (BIOPHEN Heparin LRT, Hyphen BioMed).
RESULTS
DS per se had a detectable anti-Xa effect. FXa inhibition in UFH-spiked plasma linearly increased with increasing concentrations of added DS, with a plateau at approximately 160 μg/mL DS, at which the apparent anti-Xa level had almost doubled. In the presence of heparin antagonists, the addition of DS increased anti-Xa levels, corresponding to the dissociation of the UFH-antagonists complexes . With the anti-Xa assay containing DS, UFH inhibition was not detected.
CONCLUSION
In the presence of high concentrations of DS, FXa inhibition was much higher than that predicted from added UFH amounts, presumably related to the greater availability of UFH for interaction with antithrombin. While the relevance of measuring this "masked" heparin has not been demonstrated, the presence of DS renders the result inaccurate in the presence of protamine or Polybrene.
PubMed: 38193053
DOI: 10.1016/j.rpth.2023.102257